Defining the Role of the Post-Translational Modification UFMylation in Orthoflavivirus Infection

Abstract

Orthoflaviviruses are a genus of positive strand RNA viruses that represent a significant human health burden. Orthoflaviviruses exploit host machinery, including RNA binding proteins, membrane trafficking proteins, and post-translational modification machinery to facilitate their infection. Roles for multiple post-translational modifications, including ubiquitination and glycosylation, are well described during orthoflavivirus infection, but roles for many ubiquitin-like modifications, including UFMylation, have not been examined. UFMylation describes the addition of UFM1, a ubiquitin-like protein, onto the lysine residues of target proteins, mediated by an E1 activase (UBA5), E2 conjugase (UFC1) and E3 ligase (UFL1 and UFBP1) system. UFMylation has been described to regulate infection by diverse viruses and several host pathways essential to orthoflavivirus infection, but its role in orthoflavivirus infection is unknown. Thus, we set out to examine the role of UFMylation machinery in regulation of orthoflavivirus infection. In this work, I found that orthoflaviviral infection is promoted by the UFMylation machinery. The enzymes that facilitate UFM1 activation and conjugation and the process of UFM1 conjugation promote DENV and ZIKV infectious particle production. Further, I found that UFL1 specifically regulates viruses within the orthoflavivirus genus, including DENV, ZIKV, WNV, and YFV, but not the hepacivirus HCV. Interestingly, I demonstrated that UFL1 does not alter IFN induction or response during ZIKV infection, indicating that UFMylation is regulating orthoflavivirus infection in an IFN-independent manner. Mechanistically, I examined multiple stages of viral infection using reporter virus systems and found that UFL1 does not regulate viral translation or RNA replication. Further, I examined the later stages of viral infection, and found that infectious virions within the cell, secreted infectious virions, and extracellular RNA were decreased with UFL1 depletion, indicating that UFL1 regulates a stage of viral infection following RNA replication, but prior to viral egress, suggesting that UFL1 regulates assembly of the virion. I further identified interactions between UFL1 and several viral proteins, including DENV Capsid, NS2A and NS2B-NS3 and ZIKV NS2A and NS3, via over-expression systems and during infection. Together, these results define UFMylation as a novel host-process that promotes orthoflavivirus infection.